Growth of Isolated Amastigotes of Trypanosoma cruzi in Cell-Free Medium1

Amastigotes of Trypanosoma cruzi were purified from overlays of infected Vero cell cultures by centrifugation over a discontinuous gradient of metrizamide. Pure amastigote preparations were usually recovered from the pellet under the layer of specific gravity 1.086. The isolated amastigotes grew in cell-free ML-15HA medium. Growth rate for the different strains of T. cruzi were in the order Y > Tulahuan > CL. The generation time of amastigotes in ML-15HA medium was 16.8, 18.0, and 26.4 h for the Y, Tulahuen, and CL strains, respectively, in the presence of 5% CO₂) and 16.8, 31.2, and 36.4 h, respectively, in the absence of CO₂. Intracellular amastigotes did not differ ultrastructurally from amastigotes from either the density-gradient fractionation or culture in cell-free medium.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

The interaction of myotropic and macrophagotropic strains of Trypanosoma cruzi with myoblasts and fibers of skeletal muscle

The process of interaction of bloodstream trypomastigotes from the myotropic CL and Colombiana strains and the macrophagotropic Y strain of Trypanosoma cruzi with mouse myoblasts and myotubes was analysed. After 24 h of parasite-host cell interaction, parasites from the CL and Colombiana strains appeared to be more infective to myoblasts than those from the Y strain. Parasites from the Colombiana strain were more infective for myotubes than those from the Y strain, while those from the CL strain showed very a low ability to infect the cells. For all strains the infectivity was low for short periods of interaction, increasing with time. Myoblasts infected with parasites from the Y strain fused with other infected and uninfected cells to form myotubes. However, the process of fusion was blocked when the myoblasts were infected with parasites from the CL and Colombiana strains. These data indicate a different behavior of muscle cells when in contact with myotropic or non-myotropic strains of T. cruzi.     

Trypanosoma cruzi: surface charge and freeze-fracture of amastigotes

Amastigotes of Trypanosoma cruzi, within vertebrate cells or isolated from the supernatant of vertebrate cell cultures (L-A9 fibroblast or J774G8 macrophage-like cell lines), possess glycoproteins or glycolipids on the cell surface according to the periodic acid-thiosemicarbazide-silver proteinate technique used in association with electron microscopy. The cell surface of isolated amastigotes is negatively charged, as evaluated by the binding of cationic particles (colloidal iron hydroxyde at pH 1.8 and cationized ferritin at pH 7.2) as well as by direct measurement of cellular electrophoretic mobility. Amastigotes (Y strain) isolated from the spleen of infected mice and amastigotes (Y and CL strains) from the supernatant of cell cultures previously infected with T. cruzi have the same mean electrophoretic mobility (-0.85 micron sec-1 V-1 cm). It is intermediate between the epimastigote and the trypomastigote forms (determined previously). Sialic acid is the important component responsible for the negative surface charge, as determined by the use of neuraminidase. Thus, it is possible to use the mean electrophoretic mobility as an indicator for identifying amastigotes of T. cruzi.     

Active entry of bloodstream forms of Trypanosoma cruzi into macrophages.

The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi when not able to differentiate into amastigotes. Their uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.     

Immunologically relevant strain polymorphism in the Amastigote Surface Protein 2 of Trypanosoma cruzi

Several evidences suggest that the Amastigote Surface Protein-2 (ASP-2) of Trypanosoma cruzi is an important target for immunity during infection. Based on this, we considered it important to evaluate its strain polymorphism. Initially, we observed the presence of conserved cross-reactive epitopes in amastigotes of all parasite strains tested. In addition, the predicted amino acid sequences of the genes isolated from the cDNA of amastigotes of CL-Brener, Tulahuen, Colombian and G strains displayed a high degree of identity (>80%) to the previously described genes of ASP-2. Unexpectedly, Sylvio X10/4 and G strains expressed a new isoform of ASP-2 with limited identity to the previously described genes, but with a high degree of identity when compared to each other. Immunological studies confirmed the presence of cross-reactive epitopes between recombinant proteins representing the different isoforms of ASP-2. However, the genetic vaccination of mice with the new isoform of asp-2 gene expressed by the G strain failed to provide the same degree of protective immunity to a challenge by parasites of the Y strain as did asp-2 genes of Y or CL-Brener strains. In summary, we found that few strains can express different isoforms of ASP-2 which may not share cross-protective epitopes.     

Trypanosoma cruzi invasion is associated with trogocytosis

Trogocytosis was originally thought to be restricted to the interaction of cells of the immune system with cancer cells. Such membrane exchanges are probably a general process in cell biology, and membrane exchange has been demonstrated to occur between non-immune cells within an organism. Herein, we report that membrane and protein exchange, consistent with trogocytosis, between Trypanosoma cruzi (both the Brazil and Tulahuen strains) and the mammalian cells it infects. Transfer of labeled membrane patches was monitored by labeling of either parasites or host cells, i.e. human foreskin fibroblasts and rat myoblasts. Trypomastigotes and amastigotes transferred specific surface glycoproteins to the host cells along with membranes. Exchange of membranes between the parasite and host cells occurred during successful invasion. Extracellular amastigotes did not transfer membrane patches and were did not transfer either membranes or proteins to the host cells. Membrane exchange was also found to occur between interacting epimastigotes in cell-free culture and may be important in parasite–parasite interactions as well. Further studies should provide new insights into pathogenesis and provide targets for therapeutic intervention.     

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.     

Species-specific monoclonal antibodies for a membrane antigen(s) in all developmental forms of Trypanosoma cruzi

Two monoclonal antibodies (designated as TCF48 and TCF87 were raised against Trypanosoma cruzi, strain Tulahuen, Both antibodies reacted with all developmental forms of several different strains of Trypanosoma cruzi. The antibodies showed no detectable cross-reactivity with other species of Trypanosomatidae, so far examined. TCF48 and TCF87 were classified as immunoglobulin subclasses IgG1 and IgG2b, respectively. Apparent molecular weight of the corresponding antigen(s) to these monoclonal antibodies was 25,000 in amastigotes and epimastigotes, and 25,000 and 24,000 in trypomastigotes, as determined by the Western immunoblotting analysis. This antigen appeared to be located at the plasma membrane and the flagellum ofT. cruzi. However, no evidence supported the localization of the epitope(s) at the external surface of the live cell. Since this antigen reacted with the sera from the chronically infected mice, these monoclonal antibodies may be useful in the study of Chagas' disease.     

Heme synthesis in Trypanosoma cruzi: influence of the strain and culture medium

The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in two strains of Trypanosoma cruzi (Y and CL) grown in two culture media (LIT and Warren): succinyl coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. ALA and PGB were detected in both strains of T. cruzi. However, ALA was not detected in epimastigotes of the Y strain grown in the LIT medium. The content of ALA and PBG varied according to the strain and the growth medium. No free porphyrins and heme were detected in both strains of T. cruzi. The activity of Suc.CoA-S and DOVA-T was markedly influenced by the strains of the parasite and the growth medium. No significant DOVA-T activity was detected in epimastigotes of the CL strain grown in the Warren's medium. No significant activity of ALA-D, PBGase and deaminase was detected in T. cruzi. Activity of Heme-S was detected in both strains of T. cruzi when mesoporphyrin, protoporphyrin or deuteroporphyrin was used as substrate. The enzyme activity was influenced by the strain of the parasite, the growth medium and the substrate used.     

Biological characterization of clones of the Y, CL and MR strains of Trypanosoma cruzi in inbred C3H mice

Ten clones of Trypanosoma cruzi isolated from Y, CL and MR strains were studied. The infectivity of culture forms, parasitemia pattern, polymorphism and mortality were studied in C3H inbred mice. Significant intra-group differences among Y and CL clones were found. MR clones showed higher homogeneity. These data indicate that T. cruzi strains can show different degrees of heterogeneity. It is suggested that conditions used to maintain T. cruzi strains may result in a selective advantage for some subpopulations (clones) after many years of laboratory maintenance.     

Enzymatic activity, protein expression, and gene sequence of cruzipain in virulent and attenuated Trypanosoma cruzi strains.

Protein expression, characterized in Western blots and gelatinolytic activity, of cruzipain (Cr), the major Trypanosoma cruzi cysteine proteinase, was compared among 3 attenuated T. cruzi strains (TUL 0, TCC, and Y null) and their virulent counterparts (TUL 2, Tulahuen, and Y). All attenuated strains displayed a weaker gelatinolytic activity as compared with their virulent counterparts. The electrophoretic mobility and immunological reactivity revealed quantitative and qualitative differences, with the attenuated parasites showing bands of less density in all strains and lower mobility in 2 of them, as compared with the virulent strains. Sequence analysis of 1 Cr gene in the Tulahuen and TCC strains indicated 37/1404 base pair substitutions, corresponding to 20 amino acid changes in the attenuated strain. A similar comparative analysis of 1 Cr gene between Y and Y null strains showed 13/1404 base pair substitutions, corresponding to 8 amino acid changes in the attenuated strain. Although enough variability exists in the Cr gene to allow for less- or nonfunctional isoforms of the protein, further clones should be analyzed to establish whether attenuation is regularly associated with specific sequence changes of this enzyme.     

Passive protection of mice against infection with Trypanosoma cruzi with plasma: the use of blood- and vector bug-derived trypomastigote challenge

A study was made of the protective effects of plasma (CMP) from mice convalescent from infection with Trypanosoma cruzi. A single dose of CMP was injected into mice infected with blood trypomastigotes of 1 of each of 5 strains of T. cruzi. Protection was greatest with strains BG, M1 and Y, and least with strain Peru. Strain Tulahuen was of intermediate susceptibility. The protective effect of CMP was found to be similar in mice infected by metacyclic trypomastigotes harvested from vector bugs and mice infected by blood trypomastigotes. Plasma (IMP) from mice hyperimmunized with 6 doses of a killed T. cruzi epimastigote vaccine with saponin as an adjuvant gave no protection against challenge with strain Y, although a group of mice hyperimmunized in parallel with those from which IMP was taken were strongly resistant to challenge.     

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.     

New enzyme immunoassays for specific assay and general detection of Trypanosoma cruzi, epimastigotes.

A highly specific competitive enzyme-linked immunosorbent assay for the epimastigote of Tulahuen strain was developed by using the usual 3 immunological reagents, a rabbit antiserum specific for T. cruzi, epimastigote of Tulahuen strain, beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G and the solid-phase cell fragments of the epimastigote of Tulahuen strain. A new method, the selected antibody enzyme immunoassay (SAEIA) which generally detected all strains of the epimastigote tested with the same working range, was developed by changing only the solid-phase antigen to the epimastigote of Y strain among the 3 immunological reagents. Both assays permitted us to measure accurately as little as 1,000 parasites per assay tube. Scope of the SAEIA was limited to the epimastigote. Both life-cycle forms of T. cruzi which appear in mammals, amastigote and trypomastigote, and other kinetoplastids showed low cross-reaction values by the assay. The assay principle of the new method and a preliminary study to apply the SAEIA for finding the field T. cruzi-infected insect vectors were also reported.     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

Tissue tropism of different Trypanosoma cruzi strains.

A systematic study of the distribution of intracellular parasites in the organs and tissues was performed in groups of mice inoculated with 4 different Trypanosoma cruzi strains. An extremely high parasitism of spleen, liver, and bone marrow was observed in mice inoculated with Y and Berenice strains; with CL strain, however, parasites were almost absent in those organs. Bloodstream forms apparently present differences which facilitate or prevent their uptake by macrophages from the mononuclear phagocytic system. Parasitism of the smooth muscle from hollow organs was significantly higher with ABC and Berenice strains than with Y and CL. The importance of the distribution of intracellular stages in the pathogenesis of the disease is discussed.     

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 M, these compounds displayed a strong lytic activity. It ranged from 88.4 0.6 to 99.0 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 M and 41-87 M, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 M. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 M. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.